Colloidal suspension of nanoparticles based on an amphiphilic copolymer

ABSTRACT

The invention concerns an aqueous suspension, stable in physiological medium, of nanoparticles for delivering active principles for example insulin. Said delivering particles are based on a three-block copolymer: polyethylene glycol/hydrophilic polyaminoacid/hydrophobic polyaminoacid. Said three-block copolymer particles can be associated with an active principle without denaturing it, and perform a controlled and long-term release of said active principle in vivo, thereby providing the active principle with very prolonged release. The invention also concerns the powder-form solid from which are derived the delivering particles and the preparation of said powder-form solid and said suspension of delivering particles based on three-block copolymer. The invention further concerns pharmaceutical specialities obtainable from said delivering particles filled with active principle.

[0001] The field of the present invention is that of VectorizationNanoparticles (VP), which are useful for the administration of activeprinciples (AP). These active principles are preferably medicinalproducts or nutrients for administration to an animal or human body viathe oral or nasal, vaginal, ocular, subcutaneous, intravenous,intramuscular, intradermal, intra-peritoneal, intracerebral, etc. route.In terms of chemical nature, APs that are most particularly concerned bythe invention are hydrophilic, for example proteins, glycoproteins,peptides, polysaccharides, lipopolysaccharides or polynucleotides.

[0002] The present invention more specifically relates to colloidalsuspensions of Vectorization Nanoparticles (VP), based on polyamino acidblocks and hydrophilic polymers of the type such as PolyAlkylene Glycol(PAG), preferably PolyEthylene Glycol (PEG).

[0003] The present invention is directed both toward VPs per se andtoward AP vector systems, consisting of VPs charged with AP.

[0004] The present invention also relates to pulverulent solidscomprising these VPs.

[0005] The invention also relates to processes for preparing saidcolloidal suspensions of particles charged with AP.

[0006] The combination of AP with VPs is especially directed towardmodifying their duration of action and/or conveying them to the site oftreatment and/or increasing the bioavailability of said APs. Numerouscombination techniques have already been proposed. Such techniques aredirected firstly toward allowing the AP to be transported to its site oftherapeutic action, while at the same time protecting it against attackfrom the body (hydrolysis, enzymatic digestion, etc) and, secondly,toward controlling the release of the AP on its site of action, so as tomaintain the amount available to the body at the desired level. The APsconcerned by these changes in transportation and residence in the bodyare, for example, proteins, but may also be entirely different products,organic molecules of synthetic or natural origin. The review by M. J.Humphrey (Delivery system for peptide Drugs, edited by S. Davis and L.Illum, Plenum Press, N.Y. 1986) presents the problems regardingimproving the bioavailability of APs and the advantage of systems forvectorization and controlled release.

[0007] In point of fact, two main types of system for vectorization andcontrolled release of AP are distinguished, which are characterized bythe mode of combination of the AP with the VPs, namely:

[0008] combination by adsorption, illustrated by the attached FIG. 1,and

[0009] combination by encapsulation (or by coating), illustrated by theattached FIG. 2.

[0010] Combination of the AP with the VPs by spontaneous adsorption isthe system with which the present invention is concerned. Generally,spontaneous adsorption techniques are less aggressive with respect toAPs than the techniques of combination by encapsulation, in which use isoften made of solvents and/or surfactants, and also process steps(emulsification, evaporation, distillation) that are liable to denaturethe APs, and in particular APs of protein nature (preservation of thenative secondary structure), which constitute the majority of the usualAPs targeted. In the case of a combination by adsorption, the releasetakes place by desorption.

[0011] Beyond the modes of combination/release of the AP with respect tothe VPs, the constituent materials of the VPs must have particularworking properties. Finally, the specifications that it is desired toobtain for the VPs are particularly stringent and especially comprisethe following specifications.

[0012] 1 A first specification desired for the VPs is:

[0013] firstly, that the VPs combine easily with the APs to form VP-APsystems that allow a sustained release and sustained action of the AP invivo (for example, a duration of action of at least 24 hours for anAP=insulin), and

[0014] secondly, that these VPs (which may or may not be charged withAP) form aqueous suspensions that are stable (for example for at leastseveral months), without the aid of organic solvent and/or surfactant;i.e. the VPs remain in suspension and do not flocculate.

[0015] 2 The VPs should consist of a (co)polymer which is biocompatible,which can be eliminated (by excretion) and/or which is rapidlybiodegradable into products that are not toxic for the body.

[0016] 3 It is also desired for the VPs to have a size that is smallenough to be able to undergo, in suspension in a liquid, a sterilizingfiltration with a filter whose pore diameter is less than or equal to0.2 μm.

[0017] 4 It is desirable for the VPs and the VP-AP systems to be able tobe obtained via a process that is not denaturing for the AP.

[0018] 5 The VPs should, advantageously, allow the rate of release ofthe AP to be controlled.

[0019] 6 Another important specification is that the VP-AP systems areable to constitute excellent injectable medicinal products. Thisimproved capacity for administration by injection—e.g. intravenous orintramuscular—“injectability” is characterized by:

[0020] (i) a reduced injected volume (for a given therapeutic dose),

[0021] (ii) a low viscosity.

[0022]  These two properties are satisfied when the therapeutic dose ofAP is combined with a minimum amount of VP. In other words, the VPs musthave a high degree of charging with AP.

[0023] 7. The cost intrinsic to the VPs in an injectable preparationmust be low and, in this case also, the VPs should have a high degree ofcharging with AP. In the final analysis, the small size and a highdegree of charging are major specifications desired for VPs.

[0024] 8 It is also advantageous for the polymer constituting the VPsnot to induce an immune response.

[0025] 9 Finally, it is advantageous for the VPs to have a life time inthe body which is not less than the release time desired for the AP.

[0026] The prior technical propositions (a) to (g), described below,attempted, in vain, to satisfy all these specifications.

[0027] (a) U.S. Pat. No. 5,286,495 relates to an encapsulation processby vaporization of proteins in an aqueous phase, using oppositelycharged materials, i.e.: alginate (negatively charged) and polylysine(positively charged). This manufacturing process allows particles largerthan 35 μm to be produced.

[0028] (b) Moreover, emulsion techniques are commonly used to preparemicroparticles charged with AP. For example, patent applications WO91/06286, WO 91/06287 and WO 89/08449 disclose such emulsion techniquesin which use is made of organic solvents to dissolve polymers, forexample of polylactic type. However, it was found that the solvents maybe denaturing, especially for peptide or polypeptide APs.

[0029] (c) Biocompatible VPs known as proteinoids are also known, whichwere described as early as 1970 by X. Fox and K. Dose in “MolecularEvolution and the origin of Life”, published by Marcel Dekker Inc.(1977). Thus, patent application WO 88/01213 proposes a system based ona mixture of synthetic polypeptides, the solubility of which depends onthe pH. To obtain the matrix microparticles according to said invention,they dissolve the mixture of polypeptides and then, with a change in pH,they bring about the precipitation of proteinoid particles. When theprecipitation takes place in the presence of an AP, this AP isencapsulated in the particle.

[0030] (d) Mention will also be made, as a reminder, of U.S. Pat. No.4,351,337, which relates to a different field than that of thevectorization of AP that is intrinsic to the invention. Said patentdiscloses solid implants fixed and localized in quite specific places inthe body. These implants are hollow tubes or capsules of microscopicsize (160 μm and with a length equal to 2 000 μm), consisting ofcopolymers of copoly(amino acids)—e.g. poly(glutamic-leucine) orpoly(benzyl glutamate-leucine)—obtained by copolymerization of aminoacid N-carboxyanhydride (NCA) monomers. The inclusion of an AP takesplaces by means of a technique of evaporating solvent from a mixture ofpolymer and AP. U.S. Pat. No. 4,450,150 belongs to the same family asU.S. Pat. No. 4,351,337 studied above and has essentially the samesubject. The constituent PAAs are poly(glutamic acid-ethyl glutamate)s.

[0031] (e) Patent application PCT/FR WO 97/02810 discloses a compositionfor the controlled release of active principles, including a pluralityof lamellar particles of a biodegradable polymer, which is at leastpartly crystalline (lactic acid polymer) and of an AP adsorbed onto saidparticles. In this case, the release of the active principle takes placeby desorption.

[0032] (f) Patent application PCT WO 96/29991 has as its subject matterpolyamino acid particles that are useful for vectorizing APs such asinsulin. These particles are between 10 and 500 nm in size.

[0033] The particles according to WO 96/29991 form spontaneously byplacing PAA in contact with an aqueous solution. PAAs comprise ahydrophobic block formed by neutral and hydrophobic amino acid monomersOAA (polyLeu) and a hydrophilic block formed by ionizable andhydrophilic monomers IAA (polyGlu).

[0034] (g) EP 0 583 955 discloses polymer micelles capable of physicallytrapping hydrophobic APs. These micelles consist of block copolymersincluding a hydrophilic block consisting of polyethylene glycol (PEG)and a hydrophobic block consisting of a polyamino acid, for example:PEG/polyONAA (ONAA=hydrophobic neutral amino acid).

[0035] The ONAA may be: Leu, Val, Phe, Bz-O-Glu or Bz-O-Asp, this lastamino acid being preferred. The hydrophobic AP active principles trappedin these PEG/polyONAA micelles are, for example: adriamycin,indomethacin, daunomycin, methotrexate, mitomycin.

[0036] In this patent application, the only examples presented aremicelles based on PEG/polyGlu-O-Bz. Now, it is known that these Glu-O-Bzesters are not stable to hydrolysis in aqueous medium. In addition, theenzymatic hydrolysis of these products forms potentially toxic unnaturalbenzene derivatives. Moreover, nowhere in said document is it a matterof particles consisting of a PEG/polyONAA block copolymer, the core ofwhich is formed by the hydrophobic neutral polyamino acid and comprisinghydrophilic outer hair based on PEG, these particles being capable ofcombining with hydrophilic APs and of releasing them in vivo.

[0037] It is moreover known that PEGs are not biodegradable and are evencapable of protecting VPs from enzymatic degradation, thus impairing thein vivo biodegradability of the VPs, which is one of the essentialcharacteristics desired in the context of the present invention.

[0038] It thus emerges from the foregoing text that the prior technicalpropositions described above do not completely satisfy thespecifications indicated above, and in particular specifications 1(sustained duration of release and of in vivo action and stability inaqueous suspension of the VPs), 2 (biodegradability), 4 (non-denaturingproduction), 5 (controlled rate of release) and 8 (absence of immuneresponse).

[0039] Given this state of affairs, one of the essential objectives isto be able to provide novel VPs (of the adsorption/desorption type)which spontaneously form, without the aid of surfactants or organicsolvents, stable aqueous suspensions of VPs that are suitable forvectorizing APs (especially proteins such as insulin) and which aboveall make it possible to significantly increase the duration of releaseand of action in vivo of the AP, compared with the vectorization systemsknown in the prior art and presented above (a-g, see above).

[0040] Another essential objective of the present invention is toprovide novel VPs as a colloidal aqueous suspension that is stable(especially to hydrolysis) or in pulverulent form and based onpolyalkylene glycol (PAG)/poly(amino acids) (PAA) block copolymers,these novel VPs needing to optimally satisfy specifications 1 to 9 ofthe above-targeted list of specifications.

[0041] Another essential objective of the invention is to improve uponthe particles disclosed in patent application EP 0 583 955.

[0042] Another essential objective of the invention is to provide a VPsuspension which, although having an outer crown of PEG, is,nevertheless, readily biodegradable.

[0043] Another essential objective of the invention is to provide anovel VP suspension whose characteristics are fully controlled,especially in terms of the degree of charging with AP and in terms ofcontrol of the AP release kinetics.

[0044] Another essential objective of the invention is to provide stablemedicinal VP suspensions that may be administered to man or animals, forexample via the oral or parenteral route.

[0045] Another essential objective of the invention is to provide anaqueous colloidal suspension or a pulverulent solid comprisingvectorization particles of active principles that satisfy thespecifications targeted above and that consist of a presentation formthat is appropriate and suitable for administration, for example an oraladministration, to man or animals.

[0046] Another essential objective of the invention is to propose aprocess for preparing particles (dry or as a suspension in a liquid) ofPAAs that are useful, especially, as vectors for active principles(especially proteins such as insulin), said process needing to besimpler to carry out, non-denaturing for the active principles and alsoneeding to still allow fine control of the mean particle size of theparticles obtained.

[0047] Another essential objective of the invention is the use of theabovementioned particles in aqueous suspension or in solid form for thepreparation of medicinal products (e.g. vaccines), in particular foradministration, especially oral, nasal, vaginal, ocular, subcutaneous,intravenous, intramuscular, intradermal, intraperitoneal orintracerebral administration, the hydrophilic active principles of thesemedicinal products possibly being, especially, proteins, glycoproteins,peptides, polysaccharides, lipopolysaccharides, oligonucleotides andpolynucleotides.

[0048] Another objective of the present invention is to provide amedicinal product, of the type comprising a system with sustainedrelease of active principles, which is easy and economical to produceand which is also biocompatible and capable of ensuring a very highlevel of bioavailability of the AP.

[0049] Another essential objective of the invention is to provide avaccine vectorization system, which is not immunogenic intrinsically andin combination with one or more antigens.

[0050] These objectives (among others) are achieved by the presentinvention, which relates, firstly, to a colloidal suspension ofnanoparticles that may be used especially for vectorizing activeprinciple(s) (AP(s)), these particles being individualizedsupramolecular arrangements. The particular feature of this suspensionis that said particles:

[0051] are based on at least one amphiphilic copolymer including:

[0052] at least one block of hydrophilic polymer(s) of the polyalkyleneglycol (PAG) type, preferably polyethylene glycol (PEG); and

[0053] at least one linear amphiphilic copolyamino acid (PAA),containing α-peptide chains; and

[0054] are capable of combining in colloidal suspension in undissolvedform, with at least one AP and of releasing said AP, especially in vivo,in a sustained and/or delayed manner.

[0055] One of the inventive grounds of these novel vectorizationparticles VPs, in stable colloidal aqueous suspension or in pulverulentsolid form, relates to the novel selection of an amphiphilic PAG/PAAcopolymer, for example a [hydrophilic polymer/hydrophilic polyaminoacid/hydrophobic polyamino acid] block terpolymer, allowing theproduction of particles of nanometric size (10-500 nm), which form anaqueous colloidal suspension that is stable, both with respect tohydrolysis and with respect to flocculation, in the absence ofsurfactants and/or solvents, and which can bind to APs by adsorption andrelease these APs by desorption. In the case of the present invention,the adsorption takes place naturally and spontaneously when thecolloidal particles and the AP are placed in contact in an aqueousmedium. The adsorption depends on the nature of the support (VP) and theamount of support available to the AP.

[0056] One of the major advantages of the present invention is that itleads to a VP-AP system having a significantly increased duration ofaction in vivo (for example 10 hours when AP=insulin), compared with theknown systems, and especially compared with the system described inpatent application WO 96/29991 and comprising poly(Glu)/polyLeu VPs.

[0057] Moreover, the fact that the VPs consist partly of a hydrophilicpolyamino acid offers the advantage of an easy possibility ofdegradation of the VPs by enzymatic hydrolysis, which facilitates theirremoval from the body.

[0058] Preferably, the amino acids of the amphiphilic copolyaminoacid(s) (PAA(s)) of which the particles are composed are of at least twotypes:

[0059] a first type comprising at least one hydrophilic amino acid(IAA);

[0060] a second type comprising at least one hydrophobic amino acid(OAA).

[0061] In practice, the amphiphilic copolyamino acid(s) (PAA(s)) ofwhich the particles are composed advantageously include(s) at least oneblock that is hydrophilic overall and at least one block that ishydrophobic overall.

[0062] In accordance with the invention, the structure of theamphiphilic copolymers and the nature of the IAA and OAA amino acids arechosen such that:

[0063] the polymer chains become spontaneously structured in the form ofsmall particles (VPs),

[0064] the particles form a stable colloidal suspension in water and inphysiological medium,

[0065] the VPs bind with proteins or other APs in aqueous medium (in theabsence of organic solvent and/or surfactant), via a spontaneousmechanism that is not denaturing for the AP,

[0066] the VPs release the APs from the AP-VP combination complex underphysiological conditions, and more specifically in vivo, withpharmacokinetic and pharmacodynamic profiles that are suitable for usesas medicament; the release kinetics depend on the nature of the PAG/PAAcopolymer (=polyIAA/polyOAA) that is the precursor of the VPs.

[0067] Thus, by varying the particular block structure of the copolymer,the phenomena of combination and of release of the AP can be controlledkinetically and quantitatively.

[0068] The hydrophobic fraction polyOAA participates in the aggregationof the polymer chains, which is central to the formation of the VPs.

[0069] According to one noteworthy mode of the invention:

[0070] the hydrophilic amino acid(s) (IAA) are selected from the groupcomprising:

[0071] amino acids with one or more ionizable chain(s), which is (are)at least partially ionized, preferably Glu and/or Asp and salts thereofand/or Lys; which are natural and nonnatural

[0072] and mixtures thereof;

[0073] and the hydrophobic amino acid(s) (OAA) are selected from thegroup comprising:

[0074] natural neutral amino acids, advantageously those of thesub-group: Leu, Ile, Val, Ala, Pro, Phe, and mixtures thereof;

[0075] rare or synthetic neutral amino acids, advantageously those ofthe sub-group; norleucin, norvalin, and mixtures thereof;

[0076] derivatives of polar amino acids, advantageously those of thesub-group: methyl glutamate, ethyl glutamate, benzyl aspartate,N-acetyllysine, and mixtures thereof; and

[0077] mixtures thereof.

[0078] According to one preferred embodiment of the invention, theamphiphilic copolyamino acid(s) (PAA(s)) of which the constituentamphiphilic copolymer of the particles is composed has a “block”structure.

[0079] According to one variant, this or these amphiphilic copolyaminoacid(s) (PAA(s)) may have a “random” structure, provided that it stillcomprises at least one block that is hydrophilic overall and at leastone block that is hydrophobic overall, which gives it itsamphiphilicity.

[0080] The preferred “block” amphiphilic PAA advantageously includes:

[0081] at least one block that is hydrophilic overall, consistingessentially of IAA amino acids, and having an absolute length of greaterthan or equal to 5 IAA monomers, preferably greater than or equal to 200IAA monomers and even more preferably between 10 and 50 IAA monomers,and

[0082] at least one block that is hydrophobic overall, consistingessentially of OAA amino acids, with an absolute length of greater thanor equal to 5 OAA monomers, preferably greater than or equal to 10 OAAmonomers and more preferably between 10 and 50 OAA monomers.

[0083] As regards the hydrophilic PAG—preferably PEG—it isadvantageously in the form of a block with an absolute length of greaterthan or equal to 5 monomers, preferably between 5 and 120 monomers andeven more preferably between 5 and 50 monomers. It should be noted thatthe PAG (e.g. PEG) blocks may be homopolymers or copolymers,homopolymers being more particularly appreciated.

[0084] Even more preferably, it is to the Applicant's credit to havechosen, as constituent material of the VPs, a particular class of blockterpolymer: hydrophilic polymer/hydrophilic polyamino acid/hydrophobicpolyamino acid, which are amphiphilic and charged. This amphiphilicitymakes it possible to obtain novel and surprising properties andespecially those mentioned above. Thus, the VPs according to theinvention form stable aqueous suspensions, in the absence of anysurfactant and of any organic solvent and at physiological pH values.Furthermore, by virtue of the choice of the triblock structure,comprising a hydrophilic block based on amino acids between the PAGportion and the hydrophobic portion, the VPs are readily degradable invivo via an enzymatic hydrolysis reaction. This is one of the key pointsof the terpolymer system according to the preferred embodiment of theinvention.

[0085] In addition, the VP/AP combinations form spontaneously and aboveall allow a release and thus action in vivo over very long periods (forexample 30 hours or more with a protein such as insulin). Thus, theaction time of the AP in vivo is long enough to significantly increasethe therapeutic cover and thereby improve the patient's comfort.

[0086] According to one concrete working example of the invention, theparticles consist of chains of “linear triblock” amphiphilic copolymersPEG/IAA/OAA, preferably corresponding to the following formula:

[0087] R1=H, linear or branched C₁-C₂₀ alkyl (substituted orunsubstituted), aryl, preferably benzyl (substituted or unsubstituted);

[0088] R2=NH, CO—NH, CS—NH, R8-(CH₂)_(t)-R9 with R8 and R9 chosenindependently from OCO, OCONH, NHCO, NHCONH, CONH, COO, NH, CO; t=1-6,[NHCH(R1)CO—]_(x);

[0089] R3=identical or different radicals along the chain and chosenfrom the groups defining the ionizable hydrophilic amino acids (naturalor synthetic derivatives), i.e. preferably, the groups (CH2)_(p)C₆H₄OM,(CH₂)_(P)CO₂M, (CH2)_(p)N(H_(c)R1_(d))₃X with p≧1, preferably=1 or 2; aand b are values between 0 and 3 and a+b=3; X preferably being achloride, bromide, sulfate, nitrate, hydrogen phosphate, acetate orlactate ion;

[0090] R4=identical or different radicals along the chain, chosen from Hand Me groups;

[0091] R5=identical or different radicals along the chain, chosen fromthe groups defining the hydrophobic amino acids (natural or syntheticderivatives), i.e. preferably the groups H, R1, (CH₂)_(q)C₆H₅, (CH₂)_(q)C₆H₄OR1, (CH₂)_(q) OR1, (CH₂)_(q) CO₂R1, (CH₂)_(q)CON(R1)₂, with q≧1,preferably=1 or 2;

[0092] R6=R4;

[0093] R7=H, R1CO with R1 as defined above, linear or branched C₁-C₂₀alkyl (substituted or unsubstituted), aryl, preferably benzyl(substituted or unsubstituted), C₁-C₆ hydroxyalkyl, H, —(CH₂)_(w)OH,—(CH₂)_(w)CO₂M, —(CH₂)_(w)(CHR1)_(z)OH, —(CH₂)_(w)NH₂, —(CH₂)_(y)C₆H₄OH,(CH2)yCO—N(R1)₂; R10=H, Me, (CH₂)_(v)OH., with w, z and v≧1 and M=metalor cation, typically an alkali metal such as Na, Li or K, or NH₄,R1_(a)NH_(b);

[0094] m>1; n>3.; y≧0; a+b=4.

[0095] In accordance with the invention, having a hydrophilic polyaminoacid (polyIAA) block in the copolymer constituting the VPs is aparticularly advantageous arrangement in that it improves thebiodegradability of the VPs. This polyIAA block is preferably arrangedbetween a hydrophilic polymer block and a hydrophobic polyamino acid(polyOAA) block.

[0096] The VP particles according to the invention have a mean size ofbetween 10 and 500 nm and preferably between 10 and 200 nm. For thepurposes of the invention, the terms “mean size” and “mean particlesize” mean the mean hydrodynamic diameter.

[0097] One of the advantages of the invention is that of achieving verygood control of the mean particle size of these species and theirparticle size distribution.

[0098] The control of the self-association of the polymer chains, andthus of the VP size, takes place via the polyamino acid composition, butalso, for the same composition, via the choice of a block structure andthe production process. In this manner, the particle size is extremelysmall, of the order of a few nanometers to a few tens of nanometers.

[0099] The suspension according to the invention is aqueous and stable.

[0100] The present invention is directed not only toward suspensions ofnaked particles, as defined above, but also suspensions of particlesincluding at least one active principle AP.

[0101] These particles, which may or may not be combined with an AP, areadvantageously in dispersed form in an aqueous liquid, but may also bein the form of a pulverulent solid, obtained from the PV suspension asdefined above.

[0102] The invention thus concerns, besides an aqueous colloidalsuspension of VPs, a pulverulent solid comprising VPs and obtained fromthe suspension according to the invention.

[0103] It should be noted that the nature of the distribution of thehydrophobic groups on the polymer chains may be controlled by means ofthe synthetic route selected. In this regard, there are many reactionschemes leading to the polymers selected as starting material forobtaining the VPs according to the invention.

[0104] In accordance with the invention, one particular mode forpreparing the VPs and the VP suspension is selected.

[0105] Thus, another essential subject of the invention relates to thepreparation of the selected particles (as described above), both in theform of a colloidal suspension and in the form of a pulverulent solid.The preparation process under consideration consists essentially:

[0106] insynthesizing PAG/polyIAA/polyOAA copolymers which areprecursors of the VPs and in converting them into structured VPparticles;

[0107] optionally, in purifying the particles thus produced;

[0108] optionally, in isolating these particles, preferably byconcentration, lyophilization, filtration or drying.

[0109] More specifically, the process is, firstly, a process forpreparing the abovementioned pulverulent solid formed from structurednanometric particles that may be used especially for the vectorizationof active principle(s), these particles being individualizedsupramolecular arrangements:

[0110] based on at least one amphiphilic copolymer including:

[0111] at least one hydrophobic block of linear polyamino acid(s) (PAA),containing (α-peptide chains, the hydrophobic amino acids OAA of whichthis PAA block is made being identical to or different than each other;

[0112] at least one hydrophilic block of linear polyamino acid(s) (PAA),containing (α-peptide chains, the hydrophobic amino acids IAA of whichthis PAA block is made being identical to or different than each other;and

[0113] at least one block of hydrophilic polymer(s) of the polyalkyleneglycol (PAG) type, preferably polyethylene glycol (PEG);

[0114] which are capable of combining in colloidal suspension, innondissolved form, with at least one AP and in releasing this AP,especially in vivo, in a sustained and/or delayed manner.

[0115] This process is characterized in that:

[0116] 1) at least one PAG block comprising at least one alkylene glycolmonomer is reacted with at least one hydrophilic PAA block comprising atleast one hydrophilic amino acid IAA monomer and with at least onehydrophobic PAA block including at least one hydrophobic amino acid OAAmonomer, this PAG block and these blocks each comprising at least onereactive function so as to obtain a PAG/polyIAA/polyOAA “block”amphiphilic copolymer;

[0117] 2) the PAG/polyIAA/polyOAA block amphiphilic copolymer obtainedin step 1 is transferred into a medium that is a nonsolvent for thehydrophobic fraction(s) of the amphiphilic copolymer—preferably intowater—, which leads to the spontaneous formation of vectorizationparticles (VPs);

[0118] 3) optionally, the reaction medium is dialyzed to purify theaqueous suspension of structured particles;

[0119] 4) optionally, this suspension from step 3 is concentrated;

[0120] 5) optionally, at least one active principle AP is combined withthe particles from step 2, 3 or 4;

[0121] 6) the liquid medium is removed to collect the pulverulent solidincluding the charged or uncharged particles.

[0122] For the purposes of the invention, the term “nonsolvent” meansthat the fractions of the amphiphilic copolymer under considerationhave, for example, a solubility of less than 1 g/l at 25° C. in thenonsolvent medium under consideration.

[0123] At the end of step 2, the liquid medium does not form a uniformsolution, but separates out into a dispersed phase (VP) and a phase thatis depleted in “block” amphiphilic copolymer.

[0124] The reactive functions of the PAG block(s) and of the polyOAA andpolyIAA blocks from step 1 may be amine or carboxylic acid functions. Itmay be envisaged to perform the polymerization leading to the PAGblock(s) and/or to the polyIAA hydrophilic block(s) and/or to thepolyOAA hydrophobic block(s) before, during or after the formation ofthe PAG-polyIAA and/or PAG-polyOAA and/or polyIAA-polyOAA linkage.

[0125] All these variants are within the capability of a person skilledin the art.

[0126] Preferably, in step 1:

[0127] 1.1) a copolymerization of monomers formed by N-carboxyamino acid(NCA) anhydrides of at least two different types, firstly of NCA-pIAA(“pIAA” denoting an IAA precursor) and secondly NCA-OAAs, is performed,in the presence:

[0128] of at least one polar solvent preferably chosen from the groupcomprising: N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAc), pyrrolidone; NMP being moreparticularly preferred; and

[0129] optionally of at least one cosolvent selected from aproticsolvents (preferably 1,4-dioxane) and/or proptic solvents (preferablypyrrolidone) and/or water and/or alcohols, methanol being particularlypreferred;

[0130] the pIAA repeating units of the PAA precursor copolymer of theparticles is converted into IAA repeating units, by performing ahydrolysis, preferably an acidic hydrolysis, for which an acidic aqueousphase is added to the organic medium described above;

[0131] 1.2) at least one PAG polymer block of polyalkylene glycol(preferably of PEG or PPG) is used or prepared by polymerization ofalkylene glycol monomers (preferably ethylene glycol or propyleneglycol); this PAG block being functionalized (advantageously only at oneof its ends) with a reactive group preferably chosen from the groupcomprising amines (in particular primary or secondary amines), alcoholsor thiols, activated carboxylic acids (by prior reaction withdicyclohexylcarbodiimide, carbonyldiimidazole or any other reagent knownto those skilled in the art);

[0132] 1.3) the functionalized PAG from step 2 is added to the mediumfor polymerization of the hydrophilic polyIAA and hydrophobic polyOAAblocks, before, during or after the polymerization.

[0133] Step 1.1 of the process is inspired from the known techniques forpolymerizing N-carboxy-α-amino acid (NCA) anhydrides, described, forexample, in the article “Biopolymers, 15, 1869 (1976)” and in the bookby H. R. Kricheldorf “α-amino acid-N-carboxy-anhydride and relatedheterocycles” Springer Verlag (1987).

[0134] According to one variant, during step 1.1, thepoly(OAA)-poly(IAA) copolymer obtained is precipitated—preferably inwater—and this precipitate is collected. This variant corresponds to abatchwise mode for preparing particles, in which the poly(OAA)-poly(IAA)copolymer is isolated in the form of a precipitate forming a stableintermediate product. This precipitate may, for example, be filtered,washed and dried.

[0135] Even more preferably, the NCA-pIAAs are NCAs of O-alkylatedglutamic or aspartic acid, for example NCA-Glu-O-Me, NCA-Glu-O-Et orNCA-Glu-O-Bz (ME=methyl-Et=ethyl).

[0136] More generally, the preparation of the particles may take place,for example, by addition of a nonsolvent for the hydrophobic fraction toa solution of the amphiphilic copolymer dissolved in a solvent,advantageously after synthesis of the terpolymer. The addition of thesolution of the terpolymer to a nonsolvent for the hydrophobic fractionconstitutes one variant of this process. The operation preferablyconsists in reducing the solubility of the hydrophobic fraction so thatit aggregates, doing so to form the VPs. A person skilled in the art iscapable of finding other means for reducing the solubility of thehydrophobic fraction of the polymer, for example by modifying thetemperature, the nature of the solvent(s) and of the nonsolvent, or bycombining different techniques.

[0137] For example, during this preparation of, a colloidal suspension,the PAG-poly(IAA)-poly(OAA) amphiphilic copolymers from step 1 areplaced in an aqueous medium in which at least some of the PAGs issoluble and at least some of the OAAs is insoluble. ThePAG-polyIAA-polyOAA copolymers exist in the form of nanoparticles inthis aqueous medium.

[0138] One alternative for preparing the VP suspension according to theinvention consists in placing the pulverulent solid, as described aboveas a product and by the process for obtaining it, in contact with anaqueous medium, and in particular with water, that is a nonsolvent forthe hydrophobic fraction of the amphiphilic copolymer.

[0139] Thus, the VPs may be obtained in water in the absence of anysolvent or surfactant.

[0140] Preferably, the functionalized PAG block(s) is (are) introducedbefore and/or at the start of the polymerization, which preferablyproceeds at a temperature of between 20 and 120° C. at normalatmospheric pressure.

[0141] Advantageously, the PAGs from step 1.2 are commercially availableproducts (e.g. PEG), or else are obtained in a manner that is known perse by polymerization of ethylene oxide.

[0142] Other parameters, such as the polymer concentration, thetemperature of the reaction mixture, the mode of addition of thehydrophilic polymer, the use of reduced pressure, the reaction time,etc. are adjusted according to the desired effects and are well known tothose skilled in the art.

[0143] The description of the characteristics of the polymers, givenabove in the context of the presentation of the particles, may betransposed in its entirety in the present description relating to theprocess. Thus, in accordance with the process according to theinvention, the nature and amount of the repeating amino acids, and alsothe operating conditions, may be selected so as to obtain differenttypes of polymers having the abovementioned characteristics.

[0144] To perform the combination (step 3) of one or more APs with theparticles, it is possible to perform several methods in accordance withthe invention. Nonlimiting examples of these methods are listed below.

[0145] According to a first method, AP is combined with the particles byplacing a liquid phase (aqueous or nonaqueous) containing the AP incontact with the colloidal suspension of particles.

[0146] According to a second method, the AP is combined with theparticles by placing an AP in solid form in contact with the colloidalsuspension of particles. The solid AP may be, for example, in the formof a lyophilizate, a precipitate, a powder or the like.

[0147] According to a third method, the pulverulent solid (PAA), asdescribed above as a product and by its production characteristics, isplaced in contact with a liquid phase (aqueous or nonaqueous) containingthe AP.

[0148] According to a fourth method, the pulverulent solid, as describedabove as a product and by its production characteristics, is placed incontact with the AP in solid form. This mixture of solids is thendispersed in a liquid phase, preferably an aqueous solution.

[0149] In all these methods, the AP used may be in pure or preformulatedform.

[0150] The preparation of the VPs is advantageously followed by apurification step, involving techniques known to those skilled in theart. After this optional purification step, a colloidal suspension of VPis obtained, which may be used directly, or which it may be envisaged toisolate or collect by any suitable physical means known per se, forinstance: by filtration, by concentration, by ultrafiltration, bydensity-gradient separation, by centrifugation, by precipitation,optionally by adding a salt, or by lyophilization.

[0151] In accordance with the optional step 5, the impurities (salts)and the solvent are removed by any suitable physical separationtreatment, for example by diafiltration (dialysis) (step 4), filtration,pH modification, chromatography or distillation. Such methods make itpossible to remove the unwanted salts or solvents.

[0152] To concentrate (step 6) or to separate (step 7) the structuredparticles obtained, from their liquid suspension medium, the aqueousphase is optionally removed, for example by distillation, by drying(e.g. in an oven), by lyophilization or any other suitable physicalmeans: ultrafiltration, centrifugation. After this step 7, a pulverulentwhite solid is recovered.

[0153] Given the nanometric size of the particles, the suspension may befiltered through sterilization filters, which makes it possible toobtain sterile injectable medicinal liquids, readily and cheaply. Beingable, by virtue of the invention, to control the size of the particlesand to achieve hydrodynamic diameter (Dh) values of between 25 and 100nm is an importance advantage.

[0154] The present invention is also directed toward novel intermediateproducts of the process described above, characterized in that theyconsist of PAG-polyIAA-polyOAA copolymers that are particle precursors.

[0155] According to another of its aspects, the invention relates to asuspension and/or a pulverulent solid, as defined above and/or asobtained by the process presented above, this suspension and this solidincluding at least one active principle preferably chosen from:

[0156] vaccines, taken alone or combined with at least one antigen;

[0157] proteins and/or peptides, among which the ones most preferablyselected are: hemoglobins, cytochromes, albumins, interferons, antigens,antibodies, erythropoietin, insulin, growth hormones, factors VIII andIX, interleukins or mixtures thereof, and hematopoiesis-stimulatingfactors;

[0158] polysaccharides, heparin being more particularly selected;

[0159] nucleic acids, and preferably RNA and/or DNA oligonucleotides;

[0160] nonpeptide-protein molecules belonging to various anticancerchemotherapy classes, and in particular anthracyclines and taxoids;

[0161] and mixtures thereof.

[0162] Finally, the invention relates to a pharmaceutical, nutritional,plant-protection or cosmetic speciality product, characterized in thatit includes a suspension and/or a pulverulent solid charged with AP andas defined above.

[0163] According to another of its subjects, the invention is alsodirected toward the use of these VPs (in suspension or in solid form)charged with AP, for the manufacture of medicinal products of the typesuch as systems with controlled release of AP.

[0164] They may be, for example, medicinal products that may preferablybe administered via the oral, nasal, vaginal, ocular, subcutaneous,intravenous, intramuscular, intradermal, intraperitoneal orintracerebral route

[0165] The cosmetic applications that may be envisaged are, for example,compositions comprising an AP combined with the VPs according to theinvention, which may be applied transdermally.

[0166] The examples that follow and that relate to the hydrophilic APformed by insulin will allow the invention to be understood more clearlyin its various product/process/application aspects. These examplesillustrate the preparation of polyamino acid particles optionallycharged with AP, and similarly they have the structure characteristicsand the properties of these particles.

KEY TO THE FIGURES

[0167]FIG. 1—Scheme of a vectorization particle of the type that adsorbsthe AP.

[0168]FIG. 2—Scheme of a vectorization particle of the type thatencapsulates the AP.

[0169]FIG. 3—Change in glycemia G (mean as % basal) after injection of aformulation of VP (example 5) charged with insulin to a proportion of0.6 IU/kg, as a function of time t (in hours).

[0170]FIG. 4—Photograph of VPs (example 2) by transmission electronmicroscope.

[0171]FIG. 5—Change in the glycemia (mean as % basal) and in the meaninsulinemia I (in MU/l) in dogs, as a function of time t (in hours),after injection of a VP formulation (example 6) charged with insulin toa proportion of 0.6 IU/kg

[0172]FIG. 6—Change in the glycemia G (mean as basal %) and in the meaninsulinemia I (in MU/l) in dogs, as a function of time t (in hours)after injection of a VP formulation (example 9) charged with insulin toa proportion of 0.6 IU/kg.

EXAMPLES Example 1 Preparation ofpoly(leucine)12-block-poly(glutamate)35-(polyethylene glycol)113

[0173] The techniques used for the polymerization of the NCAs intopolymers of block or random structure are known to those skilled in theart and are detailed in the book by H. R. Kricheldorf “α-AminoAcid-N-Carboxy Anhydrides and Related Heterocycles”, Springer Verlag(1987). The following synthesis specifies the synthesis of one of them.

[0174] 4.96 g of aminoethyl-PEG (molar mass 5000; Degree ofPolymerization (DP) 113) are dissolved in 120 ml of NMP at 40° C. 1.4 mlof MeOH are added thereto, followed by addition of 6.4 g of NCA-GluOMein a single portion. After half an hour, 1.8 g of NCA-Leu are added andthe reaction is continued for 2 hours. Next, dilute hydrochloric acid isadded to the reaction medium and the resulting mixture is heated at 80°C. for 24 hours. At 50° C., the medium is neutralized with 6 N sodiumhydroxide. This intermediate is dialyzed against water to remove thesmall soluble residues (solvent, salts). The purified solution isfreeze-dried to give a white powder. 80% yield.

Example 2 Preparation ofpoly(leucine)12-block-poly(glutamate)18-(polyethylene glycol)17

[0175] 2.01 g of aminoethyl-PEG (molar mass 750; DP 17) are dissolved in45 ml of NMP at 40° C. 1.5 ml of MeOH are added thereto, followed byaddition of 9 g of NCA-GluOMe in a single portion. After half an hour, 5g of NCA-Leu are added and the reaction is continued for 2 hours. Next,dilute hydrochloric acid is added to the reaction medium and theresulting mixture is heated at 80° C. for 24 hours. At 50° C., themedium is neutralized with 6 N sodium hydroxide. This intermediate isdialyzed against water to remove the small soluble residues (solvent,salts). The purified solution is freeze-dried to give a white powder.80% yield.

Example 3 Demonstration of the Nanoparticles by Light Scattering (LS)and by Transmission Electron Microscopy (TEM)

[0176] 10 mg of the copolymer obtained in example 1 or 2 are suspendedin 10 ml of water or an aqueous salt solution. This solution is thenintroduced into a Coulter granulometer (or laser diffractometer). Theresults of the particle size analysis for the various products testedare presented in table 1 below. TABLE 1 VP size measurements ExamplePolymer Size (nm) 1 PEG₁₁₃-3 (GLU)₃₅-(LEU)₁₂ 80 2 PEG₁₇-(GLU)₁₈(LEU)₁₂41

[0177] The vectorization particles VP prepared in the present example bysuspending the amphiphilic copolymer of example 2 in water are alsophotographed with a transmission electron microscope (FIG. 4 attached).

Example 4 Test of Combination of the Nanoparticles with a Protein(Insulin)

[0178] Starting with isotonic phosphate buffer solution of pH 7.4, ahuman insulin solution with a titer of 1.4 mg/ml, corresponding to 40IU/ml, is prepared. 10 mg of the amphiphilic copolymer according toexample 1 or 2 are dispersed in 1 ml of this insulin solution. Afterincubation for 15 hours at room temperature, the insulin combined withthe vectorization particles VP and the free insulin are separated bycentrifugation (60 000×g, 1 hour) and ultrafiltration (filtrationthreshold 300 000 D). The free insulin recovered in the filtrate isassayed by High Performance Liquid Chromatography or by ELISA and theamount of combined insulin is deduced therefrom by difference.

[0179] Table 2 below collates the results of the measurements of thedegrees of combination performed on different VPs. The degree ofcombination expresses the percentage of combined insulin relative to theinsulin used in a preparation with a titer of 1.4 mg/ml of insulin and10 mg/ml of VP. This value is converted into a degree of charge, whichexpresses the maximum amount of insulin in mg that can be combined with100 mg of VP. TABLE 2 Measurements of the degree of combination withinsulin for a 0.14 mg INSULIN/mg VP mixture Max. degree of chargeExample Polymer mg/100 mg VP 1 PEG₁₁₃-(GLU)₃₅-(LEU)₁₂ 10 2PEG₁₇-9(GLU)₁₈-(LEU)₁₂ 19

Example 5 Pharmacokinetics and Pharmacodynamics of VPs Charged withInsulin in the Case of Healthy Fasted Dogs

[0180] The suspension of VP charged with insulin, prepared in example 4,was injected into two dogs rendered diabetic by total pancreatectomy,and fasted from the evening of the previous day. Thoracic subcutaneousadministration of the preparation at 11 a.m. was performed at a dosageof 0.5 IU/kg of insulin per kg of live weight of the animal. The volumeadministered is between 0.18 and 0.24 ml. At time −4, −2, 0, 1, 2, 4, 6,8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 hours, 1 ml of blood iscollected by jugular puncture under vacuum onto a tube of sodiumheparinate. 30 μl of whole blood are used extemporaneously to measurethe glycemia. The tube is then centrifuged and decanted, and the plasmais stored at −20° C. for assay of the insulin. The results given in FIG.3 below show a large hypoglycemiant effect (on both animals) whichcontinues at least up to 24 hours after the injection. TABLE 3Measurements of the insulin action time (hypoglycemiant effect) in thepresence of VPs according to the invention Time for the glycemia toreturn to the basal level Example Polymer (in hours) Soluble insulin 1(without VP) 1 PEG₁₁₃-(GLU)₃₅-(LEU)₁₂ — 2 PEG₁₇-(GLU)₁₈-(LEU)₁₂ >30

[0181] This example demonstrates the non-denaturation of insulin in thepresence of VPs according to the invention.

[0182] Furthermore, this example makes it possible to demonstrate theincrease to more than 30 hours of the duration of action of insulincompared with unformulated insulin, and thus the utility of the VPs as adelay system for the controlled release of insulin. It also shows how itis possible to control the action time by means of an appropriate choiceof the hydrophobic group.

Comparative Example 6 Preparation of thepoly(leucine)40-block-(polyethylene glycol)113 Polymer

[0183] Synthesis of poly(Leu)₄₀-PEG: 10 g of NCA-Leu are dissolved in150 ml of NMP at 60° C. 5 ml of a solution of 2 g of aminoethyl-PEG (Mw5000) in 50 ml of NMP are added to the monomer in a single portion.After 2 hours, the reaction medium is poured into 1 L of water. Theprecipitate formed is filtered off, washed and dried. Yield>95%.

[0184] The precipitate is dissolved in 100 ml of trifluoroacetic acid,followed by addition thereto of 40 ml of water over a period of onehour. The suspension is then neutralized with sodium hydroxide, dialyzedagainst water to remove the salts thus formed, and freeze-dried toobtain a solid product.

Comparative Example 7 Pharmacokinetics and Pharmacodynamics of VPsCombined with Insulin, in Healthy Fasted Dogs

[0185] The VPs of example 6 are formulated and then injected intoanimals according to the protocol given in example 5. The results givenin FIG. 5 below show a hypogylcemiant effect (on both animals), whichcontinues up to 20 hours after the injection.

Example 8 Formulation Stability Comparison

[0186] The VPs of example 6, PEG-Leu₂₅ are formulated according toexample 7. After standing for one month at +4° C., the formulation formsa deposit of precipitate, which does not dissolve at 35° C., showing theinstability of this VP formulation.

[0187] The VPs of example 1(poly(leucine)12-block-poly(glutamate)35-poly(ethylene glycol-)113 areformulated according to example 7. After standing for one month at +4°C., the formulation remains transparent and does not form a deposit ofprecipitate, showing the stability of this VP formulation and thus theadvantage of a PEG-pplyIAA-polyOAA triblock copolymer over thePEG-polyOAA diblock copolymer.

Comparative Example 9 Preparation of the poly(leucine)12-block-(sodiumglutamate)35 Polymer and its Pharmacodynamic Analysis

[0188] The polymer is prepared according to example 1 with the followingmodification, the aminoethyl-PEG of Mw=5000 and of DP=113 is replacedwith an equivalent molar amount of aqueous ammonia. The VPs areisolated, formulated and injected according to the above examples(examples 2 and 5) at a rate of 50 mg of VP per 100 IU of insulin. Theresults given in FIG. 6 below show a hypoglycemiant effect (on bothanimals) which continues up to 20 hours after the injection.

[0189] Comments: TABLE 4 comparison of the in vivo action times of plaininsulin, of insulin combined with vectorization systems of the prior art(comparative examples 6 and 8) and of insulin combined with thevectorization particles according to the invention (example 2) Time forthe glycemia to return to the basal Example Polymer level (h) Solubleinsulin 1 (without VP) 2 PEG₁₇-(GLU)₁₈-(LEU)₁₂ >30 6 comp PEG₁₁₃-LEU₄₀20 8 comp (GLU)₃₅-(LEU)₁₂ 20

[0190] It emerges from this table that the system according to theinvention (example 2) has an in vivo action time that is markedly longer(+ than 30 hours versus 20 hours) than that of the systems of the priorart (WO 96/29991): examples 6 and 8.

1. A colloidal suspension of submicron particles that may be usedespecially for the vectorization of active principle(s) (AP(s)), theseparticles being individualized supramolecular arrangements,characterized in that these particles: are based on at least oneamphiphilic copolymer including: at least one block of hydrophilicpolymer(s) of the polyalkylene glycol (PAG) type, preferablypolyethylene glycol (PEG); and at least one linear amphiphiliccopolyamino acid (PAA), containing α-peptide chains; and are capable ofcombining in colloidal suspension in undissolved form, with at least oneAP and of releasing said AP, especially in vivo, in a sustained and/ordelayed manner.
 2. The suspension as claimed in claim 1, characterizedin that the amino acids of the amphiphilic copolyamino acid(s) (PAA(s))of which the particles are composed are of at least two types: a firsttype comprising at least one hydrophilic amino acid (IAA), preferablyselected from the group comprising: amino acids with one or moreionizable chain(s), which is (are) at least partially ionized,preferably Glu and/or Asp and salts thereof and/or Lys; mixturesthereof, a second type comprising at least one hydrophobic amino acid(OAA), preferably selected from the group comprising: natural neutralamino acids, advantageously those of the sub-group: Leu, Ile, Val, Ala,Pro, Phe, and mixtures thereof; rare or synthetic neutral amino acids,advantageously those of the sub-group; norleucin, norvalin, and mixturesthereof; derivatives of polar amino acids, advantageously those of thesub-group: methyl glutamate, ethyl glutamate, benzyl aspartate,N-acetyllysine, and mixtures thereof; and mixtures thereof.
 3. Thesuspension as claimed in claim 1 or 2, characterized in that theamphiphilic copolyamino acid(s) (PAA(s)) of which the particles arecomposed include(s) at least one block that is hydrophilic overall andat least one block that is hydrophobic overall.
 4. The suspension asclaimed in any one of claims 1 to 3, characterized in that theamphiphilic copolyamino acid(s) (PAA(s)) of which the particles arecomposed has (have) a “random” structure.
 5. The suspension as claimedin any one of claims 1 to 3, characterized in that the amphiphiliccopolyamino acid(s) (PAA(s)) of which the particles are composed has(have) a “block” structure.
 6. The suspension as claimed in claim 5,characterized in that the “block” amphiphilic PAA advantageouslyincludes: at least one block that is hydrophilic overall, consistingessentially of IAA amino acids, and having an absolute length of greaterthan or equal to 5 IAA monomers, preferably greater than or equal to 200IAA monomers and even more preferably between 10 and 50 IAA monomers,and at least one block that is hydrophobic overall, consistingessentially of OAA amino acids, with an absolute length of greater thanor equal to 5 OAA monomers, preferably greater than or equal to 10 OAAmonomers and more preferably between 10 and 50 OAA monomers.
 7. Thesuspension as claimed in any one of claims 1 to 6, characterized in thatthe hydrophilic PAG—preferably PEG—is advantageously in the form of ablock with an absolute length of greater than or equal to 5 monomers,preferably between 5 and 120 monomers and even more preferably between 5and 50 monomers.
 8. The suspension as claimed in any one of claims 1 to3 and 5 to 7, characterized in that the particles consist of chains of“linear triblock” copolymers PEG/IAA/OAA, preferably corresponding tothe following formula:

in which: R1=H, linear or branched C₁-C₂₀ alkyl (substituted orunsubstituted), aryl, preferably benzyl (substituted or unsubstituted);R2=NH, CO—NH, CS—NH, R8-(CH₂)_(t)-R9 with R8 and R9 chosen independentlyfrom OCO, OCONH, NHCO, NHCONH, CONH, COO, NH, CO; t=1-6,[NHCH(R1)CO—]_(x); R3=identical or different radicals along the chainand chosen from the groups defining the ionizable hydrophilic aminoacids (natural or synthetic derivatives), i.e. preferably, the groups(CH₂)_(p)C₆H₄OM, (CH₂)_(P)CO₂M, (CH₂)_(p)N(H_(c)R1_(d))₃X with p≧1,preferably=1 or 2; a and b are values between 0 and 3 and a+b=3; Xpreferably being a chloride, bromide, sulfate, nitrate, hydrogenphosphate, acetate or lactate ion; R4=identical or different radicalsalong the chain, chosen from H and Me groups; R5=identical or differentradicals along the chain, chosen from the groups defining thehydrophobic amino acids (natural or synthetic derivatives), i.e.preferably the groups H, R1, (CH₂)_(q)C₆H₅, (CH₂)_(q) C₆H₄OR1, (CH₂)_(q)OR1, (CH₂)_(q) CO₂R1, (CH₂)_(q)CON(R1)₂, with q≧1, preferably=1 or 2;R6=R4; R7=H, R1CO with R1 as defined above, linear or branched C₁-C₂₀alkyl (substituted or unsubstituted), aryl, preferably benzyl(substituted or unsubstituted), C₁-C₆ hydroxyalkyl, H, —(CH₂)_(w)OH,—(CH₂)_(w)CO₂M, —(CH₂)_(w)(CHR1)_(z)OH, —(CH₂)_(w)NH₂, —(CH₂)_(y)C₆H₄OH,(CH₂)yCO—N(R1)₂; R10=H, Me, (CH₂)_(v)OH, with w, z and v≧1 and M=metalor cation, typically an alkali metal such as Na, Li or K, or NH₄, R1NH₃;m>1; n>3; y>0.
 9. The suspension as claimed in any one of claims 1 to 8,characterized in that the vectorization particles (VPs) have a mean sizeof between 10 and 500 nm and preferably between 10 and 200 nm.
 10. Thesuspension as claimed in any one of claims 1 to 9, characterized in thatthe vectorization particles (VPs) include at least one active principle.11. The suspension as claimed in any one of claims 1 to 10,characterized in that it is aqueous and stable.
 12. A pulverulent solid,characterized in that it is obtained from the suspension as claimed inany one of claims 1 to
 11. 13. A process for preparing a pulverulentsolid obtained from the suspension as claimed in any one of claims 1 to3 and 5 to 11, characterized in that: 1) at least one PAG blockincluding at least one alkylene glycol monomer is reacted with at leastone hydrophilic PAA block comprising at least one hydrophilic amino acidIAA monomer and with at least one hydrophobic PAA block including atleast one hydrophobic amino acid OAA monomer, this PAG block and theseblocks each including at least one reactive function so as to obtain aPAG/polyIAA/polyOAA “block” amphiphilic copolymer; 2) thePAG/polyIAA/polyOAA block amphiphilic copolymer obtained in step 1 istransferred into a medium that is a nonsolvent for the hydrophobicfraction(s) of the amphiphilic copolymer—preferably into water—, whichleads to the spontaneous formation of PA vectorization particles; 3)optionally, the reaction medium is dialyzed to purify the aqueoussuspension of structured particles; 4) optionally, this suspension fromstep 3 is concentrated; 5) optionally, at least one active principle APis combined with the particles from step 2, 3 or 4; 6) the liquid mediumis removed to collect the pulverulent solid comprising the charged oruncharged particles.
 14. A process for preparing the suspension asclaimed in any one of claims 1 to 11, characterized in that thepulverulent solid as claimed in claim 12 and/or the pulverulent solidobtained by the process as claimed in claim 13 is placed in contact withan aqueous medium that is a nonsolvent for the hydrophobic fraction ofthe amphiphilic copolymer.
 15. A process for preparing the suspension asclaimed in any one of claims 1 to 11, characterized in that it includessteps 1, 2, 3, 4 and optionally 5 of the process as claimed in claim 13.16. A process for preparing the suspension as claimed in claim 10,characterized in that the AP is combined with the particles by placing aliquid phase containing said hydrophilic AP in contact with thecolloidal suspension of particles.
 17. A process for preparing thesuspension as claimed in claim 11, characterized in that the AP iscombined with the particles by placing said AP in solid form in contactwith the colloidal suspension of particles.
 18. A process for preparingthe suspension as claimed in any one of claims 1 to 11, characterized inthat the pulverulent solid as claimed in claim 12 and/or the pulverulentsolid obtained by the process as claimed in claim 13 is placed incontact with a liquid phase containing the AP.
 19. A process forpreparing the suspension as claimed in any one of claims 1 to 11,characterized in that the pulverulent solid as claimed in claim 12and/or the pulverulent solid obtained by the process as claimed in claim13 is placed in contact with the AP in solid form and in that thismixture of solids is dispersed in a liquid phase, preferably an aqueoussolution.
 20. A product that is an intermediate of the process asclaimed in claim 13, characterized in that it consists of amphiphilicPAA copolymers of the PAG/polyIAA/polyOAA “block” type, preferablyPEG/polyGlu or Asp/polyONAA, that are particle precursors.
 21. Thesuspension as claimed in any one of claims 1 to 11 and/or obtained bythe process as claimed in claim 13 and/or the pulverulent solid asclaimed in claim 12, including at least one active principle preferablychosen from: vaccines, taken alone or combined with at least oneantigen; proteins and/or peptides, among which the ones most preferablyselected are: hemoglobins, cytochromes, albumins, interferons, antigens,antibodies, erythropoietin, insulin, growth hormones, factors VIII andIX, interleukins or mixtures thereof, and hematopoiesis-stimulatingfactors; polysaccharides, heparin being more particularly selected;nucleic acids, and preferably RNA and/or DNA oligonucleotides;nonpeptide-protein molecules belonging to various anticancerchemotherapy classes, and in particular anthracyclines and taxoids; andmixtures thereof.
 22. A pharmaceutical, nutritional, plant-protection orcosmetic specialty product, characterized in that it includes asuspension as claimed in any one of claims 1 to 11 and/or obtained bythe process as claimed in claim 13 and/or the pulverulent solid asclaimed in claim 12.